Biological Control 29 (2004) 435–444

www.elsevier.com/locate/ybcon

The role of host semiochemicals in parasitoid specificity: a casestudy with Trissolcus brochymenae and Trissolcus simoni on

pentatomid bugs

Eric Conti,a,* Gianandrea Salerno,a Ferdinando Bin,a and S. Bradleigh Vinsonb

a DAPP-Entomology, University of Perugia, Borgo XX Giugno, Perugia I 06121, Italyb Department of Entomology, Texas A&M University, College Station, TX 77843, USA

Received 21 January 2003; accepted 22 August 2003

Abstract

The role of semiochemicals on host specificity of two egg parasitoid species, the European Trissolcus simoni and the American

Trissolcus brochymenae (Hymenoptera: Scelionidae), was studied in an olfactometer and in different arenas. Cues from two allo-

patric pests of cabbage, the European Eurydema ventrale and the American Murgantia histrionica (Heteroptera: Pentatomidae), and

from the polyphagous and cosmopolitan Nezara viridula were tested. Both T. simoni and T. brochymenae responded to volatile and

contact cues from their co-evolved hosts E. ventrale and M. histrionica, respectively, thus confirming the role of host semiochemicals

in host location and recognition. When cues of non-co-evolved hosts were presented, a partial ‘‘new association’’ was obtained, as T.

simoni probed and oviposited in M. histrionica eggs and some adult emergence occurred. However, this association is unlikely to

occur in the field because T. simoni did not respond to volatile cues of M. histrionica. Instead T. brochymenae partially responded to

volatile and contact cues from E. ventrale, but eggs were rarely accepted and parasitoids did not develop in this host. When N.

viridula was tested, T. simoni responded only to contact cues, whereas T. brochymenae partially responded to volatile and contact

cues, but N. viridula eggs were not suitable for development. Therefore, the N. viridula–T. brochymenae association reported from

the literature appears unreliable. Understanding the mechanisms that result in host specificity may help increase parasitoid safety

and predict their efficacy in biological control with old or new associations.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Murgantia histrionica; Eurydema ventrale; Nezara viridula; Scelionidae; Egg parasitoid; Host selection; New association

1. Introduction

Host specificity of natural enemies is an important

characteristic for development of safe and effective bi-

ological control programs, particularly when exotic

species are introduced, as these may shift to non-targetspecies causing negative environmental consequences

(van Lenteren, 1997; van Lenteren et al., 2003; Nechols

et al., 1992; Waage, 2001). Using monophagous or oli-

gophagous natural enemies will minimize the risk of

such undesired effects on non-target species (van Lent-

eren, 1997; van Lenteren et al., 2003; Nechols et al.,

1992).

* Corresponding author. Fax: +39-075-585-6039.

E-mail address: econti@unipg.it (E. Conti).

1049-9644/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.biocontrol.2003.08.009

However, the traditional definition of host specificity,

which is linked to the number of host or prey associa-

tions for a natural enemy, has an important limitation in

that knowledge on host associations is often poor and

that several host records in the literature are unreliable,

mainly because of erroneous host and/or parasitoididentifications (Gordh and Beardsley, 1999; Nechols

et al., 1992). Considering that the parasitoids� host rangeis largely determined by their host selection process

(Nechols et al., 1992; Vinson, 1998), it appears more

appropriate to relate parasitoid specificity to this pro-

cess. A solution has been proposed by using sequential

specificity tests on target and non-target species, both in

laboratory and, when possible, in field conditions (vanLenteren et al., 2003). Application of such risk assess-

ment methods is fundamental to prevent undesired

436 E. Conti et al. / Biological Control 29 (2004) 435–444

effects when planning natural enemy introductions.However, these methods do not provide in-depth ex-

planations of the mechanisms that determine host range;

e.g., they do not explain why a parasitoid will attack a

given species and not a closely related one. Under-

standing the mechanisms would allow a better predic-

tion of host ranges (Hopper, 2001). This could be

obtained, at least partially, by trying to understand the

kind of information a natural enemy needs during thehost selection process. Such a process has been divided

into a series of hierarchical steps, i.e., habitat preference,

host community location, host location, host recogni-

tion and acceptance, and among different ecological and

physiological factors involved, chemical cues play the

major role in all of these steps (Steidle and van Loon,

2002; Vet and Dicke, 1992; Vinson, 1998). Therefore,

these chemical cues could be used for the characteriza-tion of host specificity with clearly quantifiable elements.

When dealing with host–egg parasitoid associations,

the ensemble of chemical and physical characteristics of

the host egg, the substrate, the combined material and/

or organisms, and their possible interactions, has been

defined as egg ‘‘host unit’’ (Conti et al., 2003). In the

case of egg parasitoids of pentatomid bugs (Heterop-

tera: Pentatomidae), the host selection process has beeninvestigated mostly for Trissolcus basalis (Wollaston) on

Nezara viridula (L.) (Bin et al., 1993; Colazza et al.,

1999; Mattiacci et al., 1993), Trissolcus simoni (Mayr)

on Eurydema ventrale Klt. (Colazza and Bin, unpub-

lished observations) and Trissolcus brochymenae (Ash-

mead) on Murgantia histrionica Hahn (Conti et al.,

2003) (Hymenoptera: Scelionidae). During host search-

ing, these parasitoids respond both to volatile and tocontact chemicals from adults (Colazza et al., 1999;

Conti et al., 2003) and nymphs (Conti et al., 2003;

Salerno, 2000) of the host, although they are not the

target stage. In addition, it was shown that T. basalis

responds to volatiles systemically induced on plants by

N. viridula oviposition (Colazza et al., unpublished ob-

servations). Finally, both T. brochymenae (Conti et al.,

2003) and T. basalis (Conti et al., unpublished obser-vations) respond to short-range volatiles from the host

eggs. The host-induced plant volatiles and the adult and

nymph volatiles are probably used to locate the poten-

tial host community, the host egg volatiles appear to be

involved in the last phase of host location, whereas the

contact cues from adults and nymphs elicit parasitoid

arrestment and stimulate searching behavior on the

substrate. Finally, the process continues with host rec-ognition, which is mediated by contact chemicals pres-

ent in the host ovariole secretion used as an egg adhesive

(Bin et al., 1993; Conti et al., 2003). Therefore, all these

chemical cues are part of the ‘‘host unit’’ in the penta-

tomid bugs–Trissolcus spp. associations. Other charac-

ters, such as the internal composition of the eggs, have

not yet been considered.

In this paper we discuss the role in parasitoid speci-ficity of some chemical cues from such ‘‘host units’’

based on laboratory studies. Two allopatric associations

particularly suited for a comparison, the Palaearctic T.

simoni–E. ventrale and the Holarctic T. brochymenae–M.

histrionica, were chosen as basic models. The ‘‘European

harlequin bug,’’ E. ventrale, and the American harlequin

bug, M. histrionica, show several equivalences (sensu

Wiedenmann and Smith, 1997). They have similar hostplants, belonging to Cruciferae and Capparaceae, both

of which contain mustard oil glycosides (Aldrich et al.,

1996). They also have similar sizes, warning coloration,

life cycles, oviposition sites, and clustered eggs (i.e.,

normally 12 eggs in two rows) (Bonnemaison, 1952;

Panizzi et al., 2000). The European parasitoid T. simoni

is reported to be associated with at least 10 species of

pentatomid bugs including E. ventrale, whereas theAmerican T. brochymenae is known to be associated

with 11 pentatomid species, including M. histrionica,

(Salerno, 2000).

Because of the several host equivalences (Wieden-

mann and Smith, 1997), the question emerged as to

whether, if the two allopatric systems were brought into

contact, T. brochymenae and T. simoni would switch

hosts and, as a result, produce one or two new associ-ations. Therefore, both parasitoid species were tested

towards their co-evolved host and the non-co-evolved

species. In addition, a second question posed was whe-

ther these parasitoids would also shift to a more po-

lyphagous pentatomid species, not strictly associated

with Cruciferae and Capparaceae, such as the southern

green stink bug, N. viridula, which is a serious cosmo-

politan pest of more than 30 different crops (Panizziet al., 2000; Todd, 1989) and attacks plants belonging to

at least 32 families (Panizzi et al., 2000).

2. Materials and methods

2.1. Insects

The three cultures of pentatomid bugs were obtained

from field collected individuals. E. ventrale and N. viri-

dula were collected on wild and cultivated plants be-

longing to cruciferous species or to different families,

respectively, in the area of Perugia, central Italy. M.

histrionica was collected from cabbage in the Beltsville

area, Maryland. All pentatomid bugs were reared in

controlled condition chambers (25� 2 �C, 70� 10% RH,14:10 (L:D) h photoperiod), inside clear plastic food

containers (300� 195� 125-mm high) with 5-cm diam-

eter mesh-covered holes. Separate containers were used

for nymphs and adults. All stages were fed with seeds,

fruits, and vegetative parts of their preferred food.

Cabbage and broccoli (Brassica oleracea L.) were used

to feed E. ventrale and M. histrionica, sunflower seeds

E. Conti et al. / Biological Control 29 (2004) 435–444 437

(Helianthus annus L.) and French beans (Phaseolus vul-garis L.) for N. viridula. Food was changed every 2–3

days and water was provided weekly through soaked

cotton balls.

The egg parasitoids were field collected from their

hosts. T. brochymenae was obtained from M. histrionica

eggs laid on Isomeris arborea Nutt. (Capparaceae) at

San Diego, California, near the Borderfield State Park

(Dave Truesdale, personal communication). T. simoni

was collected from E. ventrale eggs laid on cabbage, at

Perugia, Italy. Both parasitoids were reared in 85-ml

glass tubes (30-mm diameter� 150-mm length) and kept

in an incubator (25� 1 �C, 80� 5% RH, 15:9 (L:D) h

photoperiod). Adult wasps were fed with a solution of

sugar (100 g), honey (10 g), yeast (10 g), benzoic acid

(1 g), and water (Safavi, 1968). Freshly laid (0–24 h old)

host eggs were exposed to parasitoid females for 48 h.Females were then removed and the eggs were stored

for incubation. After emergence, male and female

parasitoids were kept together to allow mating. For

experimental purposes, about 24 h before the assays,

2–5-day-old mated females were isolated in small glass

vials (10-mm diameter� 25-mm length), provided with

a drop of the Safavi (1968) diet, and kept in the same

incubator.

2.2. Host location: olfactometer bioassays of volatile

chemicals

Parasitoid response to volatile chemicals from pen-

tatomid bugs was evaluated in a Y-tube olfactometer

described in previous papers (Colazza et al., 1999; Conti

et al., 2003). The olfactometer consisted of a Plexiglasplate (15-mm thickness) with a Y-shaped cavity (stem

90-mm long; arms 80-mm long at 130� angle; internal

section 15� 15mm), sandwiched between two glass

plates. Medical-grade compressed air flowed through

both arms creating an air stream of 144ml/min per arm.

The flow was regulated by flowmeters and bubbled

through a water jar to humidify the air before it passed

into the olfactometer. The olfactometer was surroundedby a black fabric curtain to minimize possible cues from

the room, and was illuminated by two 22-W cool white

fluorescent tubes located above the device. The tem-

perature in the bioassay room was continuously main-

tained at �25 �C. For each experiment, one mated

pentatomid bug female in ovipositional state, i.e., with

enlarged and slightly bloated abdomens, was caged in a

small brass mesh box (25� 15mm) and placed close tothe air orifice at the end of one arm, randomly assigned,

or of both arms in the case of two-choices tests. An

empty box was placed in the control arm. Each species

(E. ventrale, M. histrionica or N. viridula) was used for a

set of 4–5 bioassays, each carried out with different

parasitoid females. After each set of trials the glass

plates and brass mesh boxes were cleaned with hexane,

acetone, ethanol, and distilled water, whereas the Plex-iglas part of the olfactometer was cleaned with a labo-

ratory detergent and rinsed with hot tap water (�90 �C,for 5min) and finally with distilled water. The possible

presence of bias between the two olfactometer arms was

evaluated by running blank tests (i.e., with empty arms).

All tests were conducted from �9:00 am to 7:00 pm.

A naive female of the parasitoid was introduced into

the Y-tube at the entrance of the stem. The parasitoid�swalking pattern was then recorded for 10min with a

monochrome CCD videocamera (Sony SSC M370 CE)

fitted with a 12.5–75mm/F 1.8 zoom lens. In order to

exclude reflected light, the olfactometer was illuminated

by infra-red light (homogenous emission of wavelengths

at 950 nm provided by 108 LEDs) from below, and the

camera lens was covered with an infrared pass filter

(Kodak Wratten filter 87�AA). Analog video signals fromthe camera were digitalized by a video frame grabber,

and digital data were processed by XBug, a video

tracking and motion analysis system developed for the

Linux operating system (Colazza et al., unpublished).

The behavioral response to volatile cues was measured

as the residence time percentage, i.e., the percentage of

total time spent in each of the olfactometer arms.

Data were analyzed with t tests for paired compari-sons and, prior to analysis, residence time percentages

were subjected to angular transformation when neces-

sary to normalize distributions (Statistica 5.1, Statsoft

Italia, 1997; Sokal and Rohlf, 1998).

2.3. Host location: open arena bioassays of contact

chemical traces

Parasitoid response to contact traces left on the

substrate by pentatomid bugs was investigated on a filter

paper open arena (380� 252mm) (Colazza et al., 1999;

Conti et al., 2003), where the parasitoids could move in

an unconstrained field. A central circular area (58mm of

diameter) in the open arena was contaminated by a host

gravid female. Uncontaminated areas were also tested as

controls. In order to contaminate the central area, thehost female was kept in place for 1 h and forced to walk

on the filter paper surface with a special device obtained

from a transparent polyethylene Petri cover connected

to a table watch. The axis of the watch mechanism was

introduced through a hole expressly made in the middle

of the cover with only the second hand being kept in

place, so that its movement forced the host to move

continuously and uniformly in the constrained area.A naive female parasitoid was then placed in the

middle of the central area, either host-contaminated or

uncontaminated, and its walking behavior was recorded.

The experiment was stopped when the female either

walked or flew out of the arena. Four to five bioassays

were conducted for each arena set. The parasitoid�swalking pattern was recorded and digitalized data were

438 E. Conti et al. / Biological Control 29 (2004) 435–444

processed as described for the olfactometer bioassays.The behavioral parameters, used to characterize the

walking patterns of the two different parasitoids in the

host-contaminated patch, were the total residence time

(s) and the tortuosity index. The latter ranges from 0 to

1, with 0 indicating a completely linear tracking and 1

the maximum tortuosity. This index was computed as

follows (Peri et al., unpublished):

T ¼ 1� mp=tl;

with T , tortuosity index; mp, maximum projection of the

track over the generic line in the plan; and tl the totallength of the track.

Data were analyzed with one-way analysis of vari-

ance (ANOVA) and with the Newmann–Keuls test for

multiple comparisons between the means. When neces-

sary, heteroscedastic data were normalized with angular

or logarithmic transformations (Statistica 5.1, Statsoft

Italia, 1997; Sokal and Rohlf, 1998).

2.4. Host recognition, acceptance, and suitability: closed

arena experiments with pentatomid bug eggs

Host recognition and acceptance behavior by T.

simoni and T. brochymenae was investigated in closed

arenas by exposing a simplified egg cluster (i.e., three

eggs) of either E. ventrale, M. histrionica or N. viridula

to a 2–5-day-old, naive, female parasitoid. Bioassayswere carried out in a multiple arena composed of a

Plexiglas plate (80� 60� 4-mm thick) with six holes

acting as single arenas (4-mm height� 13-mm diame-

ter), sandwiched between two glass plates. The eggs

were placed in the center of each arena, and tunnels

(3-mm diameter and 10-mm length) made on the ex-

ternal sides of each arena wall were used to introduce

a parasitoid female. Observations began after parasit-oid introduction and ceased after 10min if no en-

counter occurred, 15min after an encounter if there

was no recognition, or after parasitization. Only ob-

servations that led to an encounter were considered for

data analysis. Parasitoid behavior was recorded with a

JVC KY-M280 video camera using 125–75mm/F1.8

lenses and connected to a Panasonic NV-FS100HQ

video recorder. Behavioral data were then collectedand analyzed with The Observer Video-Pro Version

4.0 for Windows (Noldus Information Technology,

1997).

The suitability of pentatomid eggs for parasitoid de-

velopment was evaluated by calculating brood emer-

gence from the eggs in those cases where the female

parasitoid had oviposited, as indicated by marking be-

havior. In fact, after oviposition these parasitoids markthe host egg by rubbing their ovipositor across the egg

surface several times (Bin et al., 1993). Because this

work focused on parasitoid response to semiochemical

cues, more detailed parameters for evaluation of host

suitability, such as brood size, fecundity and behavior,were not considered.

Frequency (N) and mean durations (s) of behavioral

steps were analyzed with one-way ANOVA and the

Newmann–Keuls test for multiple comparisons, whereas

frequencies of probing or marking (% of females prob-

ing the host eggs) were analyzed with the Pearson v2 testand Goodman�s post hoc procedure for internal con-

trasts (Marascuilo and Serlin, 1988; Sokal and Rohlf,1998). When eggs were at least partially suitable for

parasitoid emergence, percentages of parasitoid emer-

gence were compared with the v2 test.

3. Results

The two parasitoid species responded to the cues oftheir co-evolved hosts, whereas they showed variable and

partial responses to the other pentatomid bug species.

3.1. Trissolcus simoni

3.1.1. Host location: parasitoid response to volatile

chemicals

Trissolcus simoni clearly responded to the olfactom-eter arm containing its co-evolved host, E. ventrale, as

indicated by a higher residence time (t ¼ 2:94; df ¼ 14;

P ¼ 0:011) compared with the control arm (Fig. 1). In-

stead, when M. histrionica or N. viridula were tested, T.

simoni did not show any response as the residence time

(M :h:: t ¼ 0:20; df ¼ 12; P ¼ 0:846; N :v:: t ¼ 1:33;df ¼ 15; P ¼ 0:203) was not different compared to the

controls. The blank experiment did not elicit any sig-nificant difference (t ¼ 1:51; df ¼ 13; P ¼ 0:156) be-

tween the two arms, indicating that no bias had affected

the olfactometer (Fig. 1).

3.1.2. Host location: parasitoid response to contact

chemical traces

The parasitoid responded with similar intensity to

areas contaminated by its co-evolved host, E. ventrale,or by M. histrionica and N. viridula, by increasing resi-

dence time (F ¼ 5:57; df ¼ 3; 75; P ¼ 0:002) compared

to the control, whereas the tortuosity (F ¼ 0:83;df ¼ 3; 75; P ¼ 0:484) of its walking pattern did not

change throughout the experiment (Fig. 2).

3.1.3. Host recognition, acceptance, and suitability:

parasitoid response to pentatomid bug eggs

Trissolcus simoni responded with similar intensity to

the eggs of E. ventrale, its co-evolved host, and M. his-

trionica, whereas it responded very poorly to N. viridula

eggs (Fig. 3). The frequency of encounters (F ¼ 3:91;df ¼ 2; 48; P ¼ 0:027) was lower with E. ventrale andM.

histrionica compared toN. viridula. Instead, frequency of

examination (F ¼ 1:55; df ¼ 2; 48; P ¼ 0:222) was not

Fig. 1. Behavioral responses of T. simoni and T. brochymenae (means� SE) to volatiles from E. ventrale, M. histrionica, and N. viridula in a Y-tube

olfactometer. cnt, control; ��, P < 0:01; �, P < 0:05; ns, not significant (t tests for paired comparisons).

E. Conti et al. / Biological Control 29 (2004) 435–444 439

significantly different although its duration (F ¼ 5:39;df ¼ 2; 44; P ¼ 0:008) was longer on E. ventrale and M.

histrionica compared to N. viridula. When staying (Fre-quency: F ¼ 0:70; df ¼ 2; 48; P ¼ 0:500. Duration:

F ¼ 0:25; df ¼ 2; 4; P ¼ 0:793) and grooming (Fre-

quency: F ¼ 8:10; df ¼ 2; 48; P ¼ 0:001. Duration:

F ¼ 0:65; df ¼ 2; 35; P ¼ 0:527) were considered, only

grooming frequency was significantly different, as it was

higher on N. viridula compared to the other two species.

These data on encounter, examination, and grooming for

T. simoni on N. viridula indicate a lower parasitoid re-sponse compared to that on E. ventrale and M. histrio-

nica, as was confirmed by probing and marking. In fact,

the percentage of probing by T. simoni (v2 ¼ 19:86,df ¼ 2, P < 0:001) was higher on E. ventrale and M.

histrionica compared to N. viridula, whereas its duration

(F ¼ 1:78; df ¼ 2; 22; P ¼ 0:123) was not significantly

different. The percentage of marking (v2 ¼ 10:88, df ¼ 2,

P ¼ 0:004) was also higher on E. ventrale and M. his-

trionica compared to N. viridula, on which it was negli-

gible (Fig. 3). Finally, the suitability of M. histrionica

eggs (50% of marked eggs) was only partial compared

with E. ventrale (77.4% of marked eggs) (v2 ¼ 4:11,df ¼ 1, P ¼ 0:042), whereas no parasitoids emerged from

N. viridula.

A synthesis of T. simoni responses to cues mediating

the host selection steps on E. ventrale, M. histrionica,and N. viridula is shown in Fig. 5.

3.2. Trissolcus brochymenae

3.2.1. Host location: parasitoid response to volatile

chemicals

Trissolcus brochymenae not only responded to vola-

tiles from its co-evolved host M. histrionica but also to

those from the other two pentatomid bug species

(Fig. 1). In fact, compared to controls, this parasitoid

exhibited a higher residence time in the olfactometer

arm containing M. histrionica (t ¼ 10:51; df ¼ 14;

P < 0:001), E. ventrale (t ¼ 2:81; df ¼ 14; P ¼ 0:014),and N. viridula (t ¼ 2:39; df ¼ 14; P ¼ 0:032). However,

when parasitoid response to M. histrionica and E.

ventrale was compared in the same olfactometer, resi-

dence time was higher (t ¼ 2:26; df ¼ 17; P ¼ 0:037) inthe arm containing M. histrionica, indicating a clear

preference for the volatiles from this co-evolved host

(Fig. 1). The blank test confirmed also for T. broc-

hymenae that no bias had affected the olfactometer, asresidence time (t ¼ 0:32; df ¼ 14; P ¼ 0:754) was similar

in the two arms (Fig. 1).

3.2.2. Host location: parasitoid response to contact

chemical traces

Trissolcus brochymenae responded to all areas con-

taminated by chemical traces from pentatomid bugs, but

with higher intensity towards its natural host M. his-

trionica (Fig. 2). Residence time (F ¼ 40:60; df ¼ 3; 131;

Fig. 2. Behavioral responses (means� SE) of T. simoni and T. brochymenae to contact traces from E. ventrale, M. histrionica, and N. viridula in an

open arena. cnt, control. Columns with the same letter are not significantly different at P < 0:05 (ANOVA, Newmann–Keuls test).

440 E. Conti et al. / Biological Control 29 (2004) 435–444

P < 0:001) was always higher on contaminated areas

compared with the control and was highest on areas

contaminated by M. histrionica. The parasitoid walking

pattern was also affected, as the tortuosity index(F ¼ 30:66; df ¼ 3; 131; P < 0:001Þ was higher on all

treatments than on the control and highest on M. his-

trionica (Fig. 2).

3.2.3. Host recognition, acceptance, and suitability:

parasitoid response to pentatomid bug eggs

Trissolcus brochymenae responded to the eggs of the

three species tested, with a clear preference for its co-evolved host, M. histrionica (Fig. 4). The frequencies

of encounters (F ¼ 4:15; df ¼ 2; 69; P ¼ 0:020) and

examinations (F ¼ 3:63; df ¼ 2; 71; P ¼ 0:031) were

lower on M. histrionica than E. ventrale and N. viri-

dula, whereas average examination duration (F ¼ 3:43;df ¼ 2; 69; P ¼ 0:038) was higher on M. histrionica

than on E. ventrale and intermediate on N. viridula.

The frequency of staying (F ¼ 4:51; df ¼ 2; 69;P ¼ 0:015) was lower on M. histrionica compared to

N. viridula and intermediate on E. ventrale, whereas its

durations (F ¼ 0:39; df ¼ 2; 26; P ¼ 0:684) did not

change among the treatments. Frequency of grooming

(F ¼ 9:44; df ¼ 2; 69; P < 0:001) was lower on M.

histrionica compared with the other two species, and

highest on E. ventrale, whereas its duration (F ¼ 2:54;df ¼ 2; 59; P ¼ 0:087) did not change. Frequencies of

probing (v2 ¼ 16:85, df ¼ 2, P < 0:001) and marking

(v2 ¼ 21:54, df ¼ 2, P < 0:001) were higher on M.

histrionica than on the other two species, although

probing duration (F ¼ 4:07; df ¼ 2; 34; P ¼ 0:026) on

M. histrionica was higher compared to E. ventrale but

similar to N. viridula (Fig. 4). However, T. broc-

hymenae emerged only from the eggs of M. histrionica

(from 85.19% of the marked eggs), whereas eggs of

the other two species tested were not suitable for

parasitoid development. Dissections of N. viridula eggsmarked by T. brochymenae showed that all parasitoids

had died as first-instar larvae.

A synthesis of T. brochymenae responses towards E.

ventrale, M. histrionica, and N. viridula is shown in

Fig. 5.

4. Discussion

Our comparative analysis of the parasitoid�s host

selection process confirms literature data on the two

co-evolved associations E. ventrale–T. simoni and M.

histrionica–T. brochymenae, as all of the host selection

steps analyzed in the laboratory provide a complete

Fig. 3. Behavioral responses (means�SE) of T. simoni to eggs of E. ventrale, M. histrionica, and N. viridula in a closed arena. The columns show

behavioral frequencies, the dots the duration of various behaviors. Data with the same letters are not significantly different at P < 0:05 (ANOVA,

Newmann–Keuls test; Pearson v2 test, Goodman�s post hoc procedure).

E. Conti et al. / Biological Control 29 (2004) 435–444 441

parasitoid response (Fig. 5). However, our analysis alsoshows that the field association N. viridula–T. broc-

hymenae, reported in the literature (Correea-Ferreira and

Moscardi, 1995; Johnson, 1984) cannot be confirmed. In

fact, the host selection sequence was not completed in

the laboratory tests due to partial parasitoid responses

to volatile and contact cues and to the lack of host egg

suitability (Fig. 5). This inconsistency in laboratory and

field data may be explained, for example, by a pooradaptation of this parasitoid to N. viridula, which is

supported by negligible field parasitism (Correea-Ferreira

and Moscardi, 1995). However, it may also depend on

some problem in the parasitoid�s taxonomic identity

(Gordh and Beardsley, 1999), or on possible behavioral

and/or physiological differences among different para-

sitoid and/or host strains.

On the other hand, our tests also show that a newassociation between the European parasitoid T. simoni

and the American bug M. histrionica may take placeunder laboratory conditions, although this association is

only partial because some steps in the host selection

process are incomplete. In detail, T. simoni completely

responds to chemical traces and to recognition cues, but

not to volatiles and it only develops partially in M.

histrionica eggs (Fig. 5). This suggests that, in the the-

oretical case of an accidental introduction, the proba-

bility of realization of such a new association in the fieldis low. A more in-depth study is currently in progress in

order to understand the level of suitability of M. his-

trionica eggs for T. simoni in terms of brood develop-

ment, size, fecundity, and behavior.

The other new association is even less probable, as

the laboratory attempt to associate the American T.

brochymenae and the European E. ventrale failed, this

species appearing unsuitable for T. brochymenae (Fig. 5).Therefore, a possible host switch between the European

Fig. 4. Behavioral responses (means� SE) of T. brochymenae to eggs of E. ventrale, M. histrionica, and N. viridula in a closed arena. The columns

show behavioral frequencies, the dots the duration of various behaviors. Data with the same letters are not significantly different at P < 0 :05

(ANOVA, Newmann–Keuls test; Pearson v2 test, Goodman�s post hoc procedure).

442 E. Conti et al. / Biological Control 29 (2004) 435–444

and the American systems would be non-reciprocal.

Finally, due to incomplete responses and lack of host

suitability, the N. viridula–T. simoni association is also

unlikely (Fig. 5). This result confirms literature data

showing that N. viridula is not among T. simoni�s hosts(Salerno, 2000).

In all of the failed laboratory association attempts,

the parasitoids partially responded to at least one step of

the host selection sequence (Fig. 5). Such partial re-

sponses could be explained by the presence of similar

chemicals, or of blends with similar compositions but

different ratios. Another possible explanation is that the

parasitoids may respond to any chemicals in comparison

to nothing, as the controls were blank air or clean paper.However, chemical identification will be necessary in

order to provide a clear explanation of such differences.

Obviously, our results cannot provide a complete

explanation of the mechanisms that determine a given

host–parasitoid association, and future experiments

should tentatively consider also other aspects of the host

unit (Conti et al., 2003), such as the possible effects of

host-induced plant volatiles (Colazza et al., unpublished

observations; Hilker et al., 2002), physical cues (Bin

et al., 1993; Conti et al., unpublished observations;Schmidt, 1991), the parasitoid�s capacity for associative

learning (Vet and Dicke, 1992; Vet et al., 1995), possible

differences among parasitoid and/or host strains (Al-

drich, 1995; Colazza and Rosi, 2001). In addition, in

order to provide a comprehensive view of the host range

in T. simoni and T. brochymenae, more pentatomid bugs

should be tested, including predaceous species, thus

allowing the evaluation of possible side effects onnon-targets.

In any case the results of our experiments, as well as

those conducted with other parasitoid species (Salerno

et al., unpublished observations), should provide a

Fig. 5. Scheme of responses of T. simoni and T. brochymenae to cues mediating different host selection steps in association tests with the European

bug E. ventrale, the American M. histrionica and the cosmopolitan N. viridula. Location (volatile): host location mediated by volatile cues. Location

(contact): host location mediated by contact traces. Recogn. accept.: egg recognition and acceptance as indicated by probing and marking,

respectively. Suitabil.: egg suitability as indicated by brood emergence.

E. Conti et al. / Biological Control 29 (2004) 435–444 443

methodology that, when combined with other ecologicaland biological data, such as life history, overwintering,

competition, and dispersal (van Lenteren et al., 2003),

may become a complete and reliable tool for evaluation

of the fundamental host range (i.e., genetically delim-

ited; sensu Nechols et al., 1992) of a given parasitoid

species, and compare it with the realized host range (i.e.,

the set of host species actually used; sensu Nechols et al.,

1992) obtained from the literature. This will also providea better understanding of the new vs. old associations

(Conti et al., 1991; Hokkanen and Pimentel, 1989) and,

when all characters of the host units (Conti et al., 2003)

involved are known, would make new associations

predictable on the basis of the equivalences or differ-

ences (Wiedenmann and Smith, 1997) of such host units.

Finally, such an approach will help towards finding a

new definition of host specificity based on the cues usedfor host selection. A specialist parasitoid would use

semiochemicals that are specific to a given host and/or

its host plant (Meiners et al., 2000), whereas a generalist

would use less specific cues that are common in different

host species.

Acknowledgments

We are grateful to Jeff Aldrich and Dave Truesdale

for providing M. histrionica and T. brochymenae,

respectively, to Joop van Lenteren for the useful dis-cussion and for critically revising this manuscript, and to

the two anonymous reviewers for their valuable com-

ments. We also thank Federica De Santis and Antonio

Caltagirone for their help in collecting data. This re-

search was financially supported by MiPAF ‘‘Progetto

speciale. Risorse genetiche di organismi utili per il mi-

glioramento di organismi di interesse agrario e per

un�agricoltura sostenibile’’, and by MIUR-COFIN 2000‘‘Analisi dei fattori che regolano le interazioni pianta-

fitofago-parassitoide per strategie innovative di con-

trollo biologico negli agroecosistemi’’.

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